An elevated plasma low density lipoprotein cholesterol level is an important risk factor for atherosclerotic cardiovascular disease, and excessive dietary cholesterol intake may contribute to such elevations. Dietary cholesterol must be absorbed from the intestine to reach the systemic circulation, but factors that govern cholesterol absorption in humans are poorly understood. Our studies examine absorption and metabolism of cholesterol, oxysterols, and phytosterols in human subjects using stable isotope tracers and negative ion electron capture (NIEC) mass spectrometry (MS). Such methods avoid radioactive exposure to subjects and achieve accurate measurement and certain identification of target analytes. GC/NIEC/MS provides an exquisitely sensitive and structurally specific tool for analyses of fluorinated derivatives of sterols employed in our studies. Development of these methods, including selection and preparation of appropriate derivatives and characterization of synth eticcholesterol tracers labeled with multiple stable isotope atoms, are important innovations that were supported by the Washington University Mass Spectrometry Resource. Descriptions of these methods are occuring at several national meetings each year.